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纯化原生卡斯核酶的简化协议,用于无DNA的基因组编辑

Margherita D'Amico¹, Flavia Angela Maria Maggiolini¹, Lucia Rosaria Forleo¹

¹CREA Council for Agricultural Research and Economics-Research Center for Viticulture and Enology, Via Casamassima 148, 70010 Turi, Italy.

Methods and protocols

|February 25, 2025

概括

我们开发了一种简化协议,用于净化CRISPR-Cas核酶,以免DNA编辑基因组. 这种方法提高了更多实验室对先进的基因编辑工具的可访问性.

科学领域:

分子生物学分子生物学
生物化学生物化学
基因编辑技术的技术

背景情况:

使用CRISPR关联 (Cas) 核酶进行无DNA基因组编辑,可提供高精度,并最大限度地减少非目标突变.
目前原生Cas蛋白的净化方法复杂,需要专门的设备,这阻碍了广泛采用.
卡斯核酶的直接输送避免了将外来DNA整合到宿主基因组中,这是治疗应用的关键优势.

研究的目的:

开发一种简化和可访问的协议,用于净化原生Cas9,Cas12RR和dCas9-VP64核酶.
为了确保高产量和纯化的Cas核酶的活性,适用于无DNA的基因组编辑应用.
为了促进实验室更广泛地获得基于CRISPR的先进基因编辑工具.

主要方法:

使用了一种简化协议,结合了亲和力和离子交换色谱.
使用最小的下游加工来保持蛋白质的完整性和活性.
纯化的核糖核蛋白复合体在体外分析了DNA点裂变效率.

主要成果:

简化协议成功产生了活性Cas9,Cas12RR和dCas9-VP64核酶.
实验室试验证实了纯化的核糖蛋白复合体对DNA点的有效裂变.
该协议是针对无DNA的基因组编辑应用程序进行优化.

结论:

这种简化净化协议显著降低了利用CRISPR技术的障碍.
它可以更广泛地访问无DNA基因组编辑工具,即使对于缺乏广泛蛋白质净化基础设施的实验室也是如此.
该方法支持精确基因编辑策略的发展和应用.

关键词:

净化气体的净化方法亲缘关系染色图学亲缘关系染色图学基因组编辑基因组编辑离子交换色谱学离子交换色谱学核蛋白是一种核蛋白.

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